GAL1 and GAL3 are paralogous signal transducers that functionally inactivate Gal80p to activate the Gal4p-dependent transcriptional activation of GAL genes in Saccharomyces cerevisiae in response to galactose. Unlike a wild-type strain, the gal3Δ strain shows delayed growth kinetics as a result of the signaling function of GAL1. The mechanism ensuring that GAL1 is eventually expressed to turn on the GAL switch in the gal3Δ strain remains a paradox. Using galactose and histidine growth complementation assays, we demonstrate that 0.3% of the gal3Δ cell population responds to galactose. This is corroborated by flow cytometry and microscopic analysis. The galactose responders and nonresponders isolated from the galactoseadapted population attain the original bimodal state and this phenotype is found to be as hard wired as a genetic trait. Computational analysis suggests that the log-normal distribution in GAL4 synthesis can lead to bimodal expression of GAL80, resulting in the bimodal expression of GAL genes. Heterozygosity at the GAL80 but not at the GAL1, GAL2 or GAL4 locus alters the extent of bimodality of the gal3Δ cell population. We suggest that the asymmetric expression pattern between GAL1 and GAL3 results in the ability of S. cerevisiae to activate the GAL pathway by conferring nongenetic heterogeneity.

SummaryIn S. cerevisiae, following the Whole Genome Duplication (WGD), GAL1-encoded galactokinase retained its signal transduction function but lost basal expression. On the other hand, its paralogue GAL3, lost kinase activity but retained its signalling function and basal expression, thus making it indispensable for the rapid induction of the S. cerevisiae GAL switch. However, a gal3Δ strain exhibits delayed growth kinetics due to the redundant signalling function of GAL1. The subfunctionalization between the paralogues GAL1 and GAL3 is due to expression divergence and is proposed to be due to the alteration in the Upstream Activating Sequences (UAS G). We demonstrate that the GAL switch becomes independent of GAL3 by altering the interaction between Gal4p and Gal80p without altering the configuration of UASG. In addition to the above, the altered switch of S. cerevisiae loses ultrasensitivity and stringent glucose repression. These changes caused an increase in fitness in the disaccharide melibiose at the expense of a decrease in fitness in galactose. The above altered features of the ScGAL switch are similar to the features of the GAL switch of K. lactis that diverged from S. cerevisiae before the WGD.

During protein synthesis dictated by the codon sequence of messenger RNA, the ribosome selects aminoacyl-tRNA (aa-tRNA) with high accuracy, the exact mechanism of which remains elusive. By using a single-molecule fluorescence resonance energy transfer method coupled with fluorescence emission anisotropy, we provide evidence of random thermal motion of tRNAs within the ribosome in nanosecond timescale that we refer to as fluctuations. Our results indicate that cognate aa-tRNA fluctuates less frequently than near-cognate. This is counterintuitive because cognate aa-tRNA is expected to fluctuate more frequently to reach the ribosomal A-site faster than near-cognate. In addition, cognate aa-tRNA occupies the same position in the ribosome as near-cognate. These results argue for a mechanism which guides cognate aa-tRNA more accurately toward the A-site as compared to near-cognate. We suggest that a basis for this mechanism is the induced fit of the 30S subunit upon cognate aa-tRNA binding. Our single-molecule fluorescence resonance energy transfer time traces also point to a mechanistic model for GTP hydrolysis on elongation factor Tu mediated by aa-tRNA.

Transfer RNA (tRNA) translocates inside the ribosome during translation. We studied the interaction strengths between the ribosome and tRNA at various stages of translocation. We utilized an optical trap to measure the mechanical force to rupture tRNA from the ribosome. We measured the rupture forces of aminoacyl tRNA or peptidyl tRNA mimic from the ribosome in a prepeptidyl transfer state, the pretranslocational state, and the posttranslocational state. In addition, we measured the interaction strength between the ribosome and aminoacyl-tRNA in presence of viomycin. Based on the interaction strengths between the ribosome and tRNA under these conditions, 1), we concluded that tRNA interaction with the 30S subunit is far more important than the interaction with the 50S subunit in the mechanism of translocation; and 2), we propose a mechanism of translocation where the ribosomal ratchet motion, with the aid of EF-G, drives tRNA translocation.

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